Stroke and vascular dementia are prevalent diseases which are both caused by multiple factors, including genetic susceptibility. In large part, the genetics of these disorders is complex. However, in families with CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), premature stroke and vascular dementia are caused by defects in the Notch3 gene. Our broad goal is to identify the molecular pathways governing the pathogenesis of CADASIL, which will help shed light on more common forms of stroke and dementia. We have proposed that mutant Notch3, expressed in arterial smooth muscle, assumes structural changes, allowing interactions with proteins that foster protein aggregation and toxic gain of function. In this study, we will identify and characterize proteins which interact with Notch3. In preliminary experiments, we have identified vascular gene products which interact with the Notch3 extracellular domain (the site of all CADASIL mutations) in yeast. We plan to perform exploratory experiments to characterize the potential role of these PUtative NotCH3 interactors (PUNCH) in CADASIL. In Specific Aim 1, we will demonstrate that the PUNCH proteins physically bind to Notch3 in mammalian cells; we will determine if coexpression of PUNCH proteins (with Notch3) functionally causes cell death or increases the levels of Notch3 in transfected cell lines; these features are seen pathologically in arteries of CADASIL patients. In Specific Aim 2, we will use novel transgenic mice expressing epitope tagged Notch3 extracellular domain to determine whether PUNCH and Notch3 interact in vivo; we will also examine transgenic mouse and CADASIL tissues for pathological accumulation of PUNCH. Completion of these Aims will confirm the in vitro function of PUNCH proteins in the context of Notch3 mutations and may identify new markers and biochemical pathways specific for this disease. The discovery of new Notch3 interacting proteins will also faciliate the production of new cellular and transgenic models of stroke and vascular dementia, which is one of the goals of the RFA to which we respond. In lay language, this application is designed to investigate the molecules involved in a disease, CADASIL that causes stroke and dementia. An understanding of CADASIL may provide clues to designing better treatments and prevention strategies for stroke and dementia. What is learned from this study may also provide information that will be useful for the treatment of other vascular diseases. Stroke and vascular dementia are common and debilitating diseases which are both caused by multiple factors, including genetic susceptibility. We propose to study a genetic cause of stroke and vascular dementia: CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy). Understanding CADASIL may provide clues to designing better treatments and prevention strategies for stroke and dementia. [unreadable] [unreadable] [unreadable]